1. Field of the Invention
The present invention relates to the absolute characterization of microscopic particles in solution. More particularly, the present invention relates to the absolute characterization of microscopic particles, such as polymers and colloids using static light scattering (SLS) and time-dependent static light scattering (TDSLS). In principle, the size range of detectability should run from about 20 Angstroms to 100 microns, with useful measurability in the range from 20 Angstroms to 2 microns, and a preferred range from about 20 Angstroms to 5000 Angstroms. Stated in terms of molar mass, the detectable range of particles should run from about 500 g/mole to 10.sup.14 g/mole, with useful measurability in the range of 500 g/mole to 10.sup.9 g/mole, with a preferred range from about 1000 g/mole to 10.sup.7 g/mole.
The preferred use of this invention is the determination of average, particle masses, static dimensions, interaction coefficients, and other properties, as well as their changes in time, when scattering is from a very large number of particles. This is to be distinguished from turbidometric and nephelometric techniques, in which turbidity or relative scattering of solutions is measured and compared to relative reference solutions, in order to obtain concentration of particles. The SLS technique employed refers to absolute macromolecular characterization, and not to determinations of concentrations of particulates with respect to specific relative calibration, etc. This is also to )e distinguished from devices which count and characterize single particles, although the present invention can count and characterize single particles, in addition to making SLS measurement. The least number of particles whose scattered light would be detected in the scattering volume (the volume of illuminated sample whose scattering is measured by a given photodetector) would be or the order of 20 and the maximum on the order of 4.times.10.sup.17, with the preferred range being from about 15,000 to 1.5.times.10.sup.13 particles. In terms of concentration of solute (dissolved polymer or colloid) the range would be from about 10.sup.-8 g/cm.sup.3 (for very large particles) to 0.2 g/cm.sup.3 (for very small particles) with the preferred range being from about 10.sup.-6 to 10.sup.-1 g/cm.sup.3. It should be pointed out that SLS in the absolute mode requires optically transparent solutions in which single, not multiple, scattering, dominates. Many particle concentration detectors actually work in turbid, solutions, which is a different range of conditions entirely.
SLS has proven to be a useful technique not only for characterizing equilibrium properties of microscopic particles, such as molar mass, dimensions and interactions, but also for following) time-dependent processes such as polymerization, degradation and aggregation. Measuring the time-independent angular distribution and absolute intensity of scattered light in the equilibrium cases allows the former properties to be determined, according to procedures set forth by Lord Rayleigh, Debye, Zimm and others (e.g. ref. 1). In particular, this invention can be used is conjunction with the well known procedure of Zimm to determine weight average molar mass M.sub.w, z-average mean square radius of gyration &lt;S.sup.2 &gt;.sub.z and second virial coefficient A.sub.2. Measuring the time-dependent changes in the scattered intensity allows calculation of kinetic rate constants, as well as deduction of kinetic mechanisms and particle structural features (e.g. ref. 2,3). TDSLS can be used to monitor polymerization and degradation reactions, aggregation, gelling and phase separation phenomena (e.g. ref 4).
In addition to absolute SLS and TDSLS measurements, the present invention can also simultaneously count and characterize individual particles which are much larger than the principal polymer or colloid particles; e.g., the large particles may have a radius of 5 microns, whereas the polymer may have an effective radius of 0.1 micron. The large particles may represent a contaminant or impurity, or may be an integral part of the solution, e.g., bacteria (large particles) produce a desired polymer (e.g., a polysaccharide) in a biotechnology reactor. The number density of bacteria can be followed in time, and the absolute macromolecular characterization of the polysaccharide could also be made (an auxiliary concentration detector would also be necessary if the polysaccharide concentration changes in time).
2. General Background of the Invention
SLS is currently used for three main purposes in academic, medical and industrial research and development, and industrial quality control; 1) to characterize useful averages of mass, mean square radius of gyration and second virial coefficient for unfractionated particles in equilibrium, using traditional procedures, 2) to characterize heterogeneous populations of particles which have been fractionated by techniques such as size exclusion chromatography and 3) to follow time-dependent processes. As examples of each purpose: 1) A new biological macromolecule or microstructure is isolated, or a new polymer is synthesized, and its average macromolecular characteristics are determined by SLS. A manufacturer of synthetic polymers, for water treatment, paints, coatings, adhesives, etc., would use SLS for quality control of their product. 2) A synthetic or biological polymer sample contains a wide variety of molar masses (polydispersity), and it is desirable to determine the mass and dimension distribution using a fractionation technique coupled to SLS. The purpose of this can be for fundamental research into a biological mechanism, to aid development of new products, to establish quality control specifications of new products, or tc assess the effects of different chemical or physical treatments on the product, etc. 3) It is desired to determine how quickly a polymer degrades under attack by such agents as enzymes, heat, radiation, ultrasound, etc., and this can be determined by TDSLS. This will guide studies in developing new pharmacological inhibitors or promoters, or resistant plastics, or biodegradable materials. A central problem of great economic interest, in which TDSLS can be used is to have an on-line method for determination of the build up of molecular weight during industrial polymerization processes. A further use of the present invention will be in the simultaneous measurement of SLS and particle counting in heterogenous solutions.
Clearly, SLS and TDSLS have a wide range of applications, including, but by no means limited to products such as pharmaceuticals, foodstuffs, resins, plastics, coatings, inks, adhesives, liposomes, cosmetics, water treatment and paper making chemicals, paints, additives, plasticizers, microencapsulation structures, etc.
Current technology generally consists of a transparent, hollow sample cell, usually of glass or quartz, into which a scattering sample is introduced and through which a light beam (usually from a laser) is passed. The scattered light then passes through the walls of the transparent cell, where photodetectors or fiber optic pick-ups are placed. The signal from the detected scattered light is then processed and the properties of the scattering sample deduced. Such systems require that sample be introduced into the cell, remote from the main sample batch itself. A disadvantage of a transparent sample cell is that it creates interfaces between incident light and the sample which produce unwanted stray light or `glare`. This stray light or glare, constitutes one of the major pitfalls and nuisances in the actual practice of SLS. Such cells are also relatively expensive and require fairly precise alignment for proper performance.
The present invention includes a (preferably miniaturized) submersible probe which can be brought into the sample, rather than vice versa. No transparent cell need intervene between the sample solution, the incident beam and the optical detectors. This reduces the SLS instrument to a small probe, which can be thought of now as a simple lab probe, like that of a pH or conductivity meter, to be used simply and routinely. The probe portion is relatively inexpensive to fabricate, easy to align, and can even be made to be disposable. The photodetectors, signal processing, etc. are normally remote from the probe. It is anticipated that such an SLS/TDSLS probe is substantially more economical, versatile and easy to use than currently available systems. The present invention can be used in a variety of modes for both time-independent and time-dependent measurements; 1) Submersible mode, in which the probe is submerged in a vessel containing sample solution, such as a beaker, test tube, vat, reactor, etc. 2) Fill mode, in which small amounts of sample liquid (about 3 microliters to 30 milliliters, for example) can be simply pipetted, scooped, or otherwise transferred into the probe body. 3) Flow mode, in which by means of an integrally flanged pump, or hydraulic connection to a tube with flowing sample, the sample liquid flows through the invention. This can be used for unfractionated samples, including those undergoing time-dependent processes (polymerization, degradation, cross-linking, etc.), or samples solutions fractionated by SEC or other means. 4) Insert mode in which a standard glass vial or cell containing sample liquid is inserted into the probe body, instead of filling the probe body, immersing it, or flowing the sample through it. This would be used instead of any of the other three modes, when, for example, the sample may be deleterious for the chamber, because of causticity, gelling, precipitation, etc. It can also be convenient and valuable when many samples are independently prepared and are to be measured separately, and/or when the state of prepared samples in sealed cells is to be checked periodically, without disturbing the sample due to flow, pipetting, etc. The demands of a wide variety of users can be satisfied by simply changing low cost, optical probe assembly, since the detection, electronics, computer interfacing and basic software are the same.
Brief Comparison with other Light Scattering Devices
The present invention is distinct from other light scattering devices. For example: U.S. Pat. No. 4,616,927 (Phillips, Reece and Wyatt) and U.S. Pat. No. 5,305,073 (Ford) describe the use of highly polished, optically transparent cell for absolute light scattering measurements. The current invention requires no optically transparent cell. Neither of these inventions are submersible, nor can either be considered as a `probe` which can go into the sample liquid being measured. Neither, hence, can fulfill the probe function of the current invention, and both are also less versatile, and more costly in general. A submersible light scattering probe is presented by U.S. Pat. No. 5,350,922 (Bartz), but is designed for relative measurements of fairly turbid media (e.g., muds in suspension in water). They collect scattered light, indiscriminately, from 0 to 180.degree. scattering angles, and hence cannot perform absolute light scattering on samples requiring exact specification of the scattering angle (their system could work for Rayleigh scatterers, i.e. for sizes much smaller than the incident radiation wavelength; since they are chiefly looking at particulates in suspension, which are generally very large, this condition would not be expected to be met). Furthermore, their device does interpose an optically transparent medium between the light source and the sample liquid and the detector and the sample liquid. In addition, that device cannot be used in either `fill mode` nor flow mode, and hence is also considerably less versatile. None of these inventions mentioned have the versatility and interchangeability of the present invention.
The use of simultaneous multi-angle detection is shown in U.S. Pat. No. 3,850,525, "Simultaneous Multiple Measurements in Laser Photometers".
The following patents documents are incorporated herein by reference:
U.S. Pat. Nos. 3,850,525; 3,954,342; 4,265,535; 4,363,551; 4,548,500; 4,616,927; 4,995,514; 5,129,723; 5,155,549; 5,235,179; 5,305,073; 5,350,922; 5,434,667; 5,638,174; and Great Britain Patent Application No. 2166234.